1. Field of Invention
The current invention concerns a method for in vivo or ex vivo targeted degradation of intracellular proteins in situ. In particular, the invention concerns the method for degradation of intracellular proteins by inducing in cells the production of a dual-function protein containing a domain that directs a selective degradation of targeted proteins to which it is attached as well as a domain that acts as a linker between the dual-function protein and the target protein. The protein degradation directing domain is a subregion within 97 amino acids which corresponds to the N-terminus of protein antizyme (NAZ). The invention further concerns a method for inducing in vivo or ex vivo in cells the production of the dual-function protein consisting of NAZ and linker directed to destruction of specific cellular target proteins.
2. Background and the Related Art
The body produces a large number of proteins which direct and regulate numerous physiological functions. The rapid turnover of these proteins is important in regulating cell growth and metabolism. These proteins are, under normal circumstances, strictly regulated by physiological feedback, which assures that when the level of certain protein in the body reaches desired level, a regulatory mechanism normally present in the body takes over the control of that particular protein production and either decreases it to the level which is sufficient for a normal physiological function of that protein or temporarily terminates its production.
In many instances, however, due to some pathological or pathophysiological conditions, such endogenous control is disturbed and overproduction or underproduction of certain proteins occurs.
Underproduction of these proteins is typically corrected by providing substitute proteins or drugs which would stimulate the protein production.
In case of overproduction of certain proteins, such as, for example, hormones, the problem is much more serious and typically can be corrected only by inhibiting the production of these proteins by drugs or synthetic enzymatic inhibitors. Very often these drugs are not overly selective for the particular protein but will also affect, that is inhibit, the production of other proteins. Additionally, when administered systemically, these inhibitors have often undesirable secondary systemic symptoms.
It would be, therefore, advantageous to have available a method for in vivo or ex vivo induction of selective destruction of targeted proteins in situ.
Proteins that turn over rapidly in the body are of special interest because they can quickly adjust their abundance in response to changes in synthesis or degradation. Some labile proteins have been found to interact with a second protein that promotes their degradation. Examples of these proteins are lysosome-degraded proteins which interact with 70-kD heat shock protein and tumor suppressor p53 which interacts with viral oncoproteins (Science, 346:382-385 (1989) and Cell, 67:547-556 (1991)).
Several methods are presently under consideration as modifiers of protein degradation. The first approach uses inhibitors of the proteasome or other viral or cellular proteases. These methods lack substrate specificity and in general modify the action of the proteolytic machinery rather than its action on specific substrates.
The second approach utilizes ubiquitination. Ubiquitination is a natural process introducing a modification consisting of the attachment of multiple copies of the protein ubiquitin that triggers proteolysis of many short-lived proteins (Nature, 357: 375-379 (1992); Microbiol. Rev., 56: 592-621 (1992); Ann. Rev. Biochem., 61: 761-807 (1992); J. Biol. Chem., 268: 6065-6068 (1993).
Attempts were made previously to provide a method for specific proteolytic removal of proteins via the ubiquitin-dependent proteolytic pathway. This method is, however, complicated, laborious and not suitable for clinical use. Degradation via the ubiquitin pathway requires the prior attachment of multiple ubiquitins to the target proteins. This attachment is accomplished by a family of enzymes designated ubiquitin-conjugating enzymes (E2), some of which use domains near their C-termini for target recognition. For example, PNAS, 92: 9117-9121 (1995) describes a method for modified ubiquitination by generating chimeric ubiquitin-conjugating enzymes that recognize and ubiquitinate their binding partners with high specificity in vitro. This method comprises forming a fusion containing a linker and one of the enzymes that participates in ubiquitination, thus ubiquitinating specific proteins and targeting them for destruction.
The method described in PNAS, above, however, depends on fusion maintaining enzymatic activity of E2 in general and particularly for the intended target. Multiple ubiquitins must be transferred to a substrate to cause its degradation and additional enzymes collectively called E3 may be needed for substrate ubiquitination. So far, this method has only been tested in vitro and its effectiveness and substrate specificity in vivo have not been demonstrated.
The selective targeted protein degradation method alternative to ubiquitination has not been previously described but would be very useful in controlling excess production and/or inhibition of production of certain proteins or their end-product compounds by way of selective degradation of targeted proteins. Such degradation in vivo would be useful in controlling diseases caused by the overproduction of certain proteins or compounds. Examples of such diseases are hormonal disorders, carcinogenic growth, viral, bacterial or parasitic infections, etc.
It would therefore, be advantageous to provide a method for in vivo or ex vivo substrate specific and targeted degradation of proteins in situ which method would be effective in vivo, in which the need for ubiquitination would be eliminated and the whole degradation process would be simplified by attaching a protein element that provides a functional alternative to ubiquitination to target proteins.
Physiological feedback control over protein production or termination of such production has been known and studied, for example, on polyamine biosynthetic enzymes. Polyamines presence in the body is essential for cell growth and proliferation. The key enzyme in the biosynthesis of the polyamines, ornithine decarboxylase (ODC), is highly regulated. Mammalian ODC is also one of the group of the most short lived of proteins. Its turnover can proceed along two different metabolic pathways: constitutive pathway and polyamine dependent pathway. The activity of the ODC enzyme is increased by cell growth in cancer but is decreased by the presence of an excess of polyamines. However, polyamine-promoted degradation of ODC seems to involve another protein.
Feedback regulation of the ODC enzyme by polyamines occurs via induction of a protein called antizyme. (Biochem. Biophys. Acta, 428: 456-465 (1976); PNAS, 73:1858-1862 (1976); J. Biol. Chem., 259:10036-10040 (1984); J. Biochem., 108:365-371 (1990) and Gene, 113:191-197 (1992)).
In their previous research, inventors discovered that regulated degradation of ODC requires interaction of two ODC regions with a specific polyamine-inducible protein antizyme. These regions are located near C-terminus and near N-terminus of ODC. This specific polyamine-inducible antizyme was found to bind to ODC, inhibit its activity and accelerate its degradation. The antizyme-ODC binding seems to be required for regulated degradation of ODC (Mol. Cell. Biol., 12: 3556-3562 (1992)). The interaction of the ODC antizyme with a ODC binding element located near the N-terminus of ODC was found essential but not sufficient for regulation of the enzyme by polyamines. The second ODC element present at the C-terminus of ODC is required for the degradation process. Thus, both the C-terminal degradation region and a binding element near the N-terminus of ODC are required for the degradation process of ODC (Mol. Cell. Biol., 13: 2377-2383 (1993)).
In the studies described in (Mol. Cell. Biol., 14: 87-92 (1994) selective degradation of ODC was found to be mediated by antizyme which binds to a region near the ODC N-terminus. This interaction induces a conformational change in ODC that exposes its C-terminus and inactivates the ODC. Additionally, it was found that while the C-terminal half of antizyme alone can bind to and inactivate ODC and alter its conformation, it cannot direct degradation of the enzyme either in vitro or in vivo. A portion of the N-terminal half of antizyme must be present to promote such degradation.
While the above findings concerning the degradation of ODC are scientifically interesting, they are without practical utility. The ODC is closely regulated and as a short life enzyme, it is quickly disposed of. Other than scientific importance and the invention background, the findings described above do not provide a method or means for selective and targeted degradation of proteins intracellularly which would have general utility.
It is, therefore, a primary objective of this invention to provide a method and means for selective and targeted degradation of proteins in situ by administering to a subject in need thereof a dual-function protein produced ex vivo, or by inducing in vivo in the subject in need of such treatment, the endogenous production of an effective amount of dual-function protein comprising a linker domain for specific binding to a target protein as well as a N-terminus domain of antizyme capable, when complexed with the target protein via the linker, of destabilizing it and changing it in such a way that it becomes labile and subject to destruction.
All patents, patent application and publication cited herein are hereby incorporated by reference.